Specific Aim 1. Retrograde Transport Regulates Autophagy-Lysosomal Function. Maintaining cellular homeostasis in neurons depends on efficient intracellular transport. Late endocytic trafficking, which delivers target materials into lysosomes, is critical for maintaining neuronal degradation capacities via autophagy-lysosomal pathways. Dynein motor-mediated retrograde transport can enhance late endocytic trafficking from distal processes to the soma, where lysosomes are predominantly localized, and drive late endosomes and lysosomes close enough to fuse with higher efficiency, thus ensuring proper autophagy-lysosomal function. Our recent study uncovered a critical role for Snapin in regulating late endocytic transport and membrane trafficking. Snapin acts as a motor adaptor by attaching dynein to late endosomes. Snapin (-/-) neurons exhibit aberrant accumulation of immature lysosomes, impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis, and impaired clearance of autolysosomes. Snapin deficiency leads to reduced neuron viability, axonal degeneration and developmental defects in the central nervous system. Reintroducing the snapin transgene rescues these phenotypes by enhancing the delivery of endosomal cargos to lysosomes and by facilitating autophagy-lysosomal function. Our studies elucidate a new mechanism that regulates neuronal autophagy-lysosomal function by coordinating two dynamic cellular processes: dynein-mediated late endocytic transport and endosomal-lysosomal membrane trafficking. Such a mechanism is critical for cellular homeostasis and essential for neuronal survival. Autophagy-lysosomal dysfunction is one of the cellular defects contributing to the pathogenesis of neurodegenerative diseases associated with accumulation of aggregation-prone proteins and damaged mitochondria. Snapin-deficient mice provide an important genetic tool for characterizing the role of impaired autophagy-lysosomal function in the clearance of aggregated proteins and dysfunctional organelles during neurodegeneration. Specific Aim 2. Regulation of Synaptic Activity by the Endolysosomal System. Proper regulation of synaptic vesicle (SV) pool size is critical to maintain synaptic activity. Two recycling pathways for SV retrieval from the plasma membrane have been described: SVs are retrieved (1) directly through clathrin-mediated endocytosis and the AP-2 complex; (2) indirectly through an early endosomal AP-3-dependent sorting mechanism; the latter has been receiving strong support from recent literature. Because early endosomes represent crossroads between local SV recycling and the endolysosomal degradation system, this raises a fundamental question: Does early endosomal sorting and SV diversion into the endolysosomal pathway have any impact on SV pool size and synaptic activity? To address this question, we applied snapin dominant-negative mutants as molecular tools combined with dual-channel time-lapse imaging in live cortical neurons. Our study reveals that dynein-driven late endosome transport regulates the SV releasable pool size. We demonstrate that the snapin-dynein (motor-adaptor) complex drives SV transport along the late endosome-lysosomal trafficking route in axons. SV components are co-transported with late endosomes along axons. Snapin-deficient neurons displayed enlarged terminals in which various degradative organelles are retained and SVs are accumulated. Elevated expression of wild-type snapin decrease the total SV pool size by facilitating SV material transport along the endolysosomal pathway. Conversely, expressing snapin mutant defective in the dynein-binding induces SV accumulation at terminals. These results provide a mechanistic link between the maintenance of SV pool size and the endolysosomal transport driven by the snapin-dynein motor-adaptor complex. Specific Aim 3. Anterograde Axonal Transport Regulates Synaptic Formation and Plasticity. The formation of new synapses and remodeling of existing synapses play an important role in the various forms of synaptic plasticity and require the targeted delivery of newly synthesized synaptic cargoes from the soma to the synaptic terminals. Thus, efficient axonal transport of newly synthesized synaptic cargoes to nascent presynaptic terminals is critical in response to neuronal activity in developing neurons. Our previous studies established that syntabulin is an adaptor capable of linking KIF5 motor and synaptic protein cargoes. Syntabulin-KIF5 mediates axonal transport of synaptic components essential for presynaptic assembly. Syntabulin loss-of-function blocks formation of new presynaptic terminals in developing neurons. Our studies establish that kinesin-mediated anterograde axonal transport is one critical factor in the cellular mechanism underlying activity-dependent presynaptic plasticity. Our study further demonstrated the critical role of syntabulin in the maintenance of presynaptic function and regulation of synaptic plasticity in well-matured sympathetic SCG neurons. Conditional syntabulin knockout mice have been recently generated in the lab. We will use this mouse line to (1) determine whether deficiency in syntabulin-mediated transport has any impact on synapse formation, maintenance and plasticity; (2) determine whether the motor-adaptor complex regulates the transport in response to synaptic activity; (3) identify the sorting signals for the axon-targeted delivery of presynaptic cargoes.